The herpes viruses include the herpes simplex viruses, comprising two related variants designated types 1 (HSV-1) and 2 (HSV-2). Many of the counterpart gene products of HSV-1 and HSV-2 have similar molecular weights and common antigenic determinants. However, clinical manifestations of HSV-1 and HSV-2 differ significantly. Nongenital herpes infections such as common cold sores are primarily caused by HSV-1. Genital and neonatal infections are most usually associated with HSV-2. About 90% of primary genital HSV infections and about 99% of recurrent genital HSV infections are caused by HSV-2. [Sullender et al., J. Infect. Dis., 157(1):164-171 (January 1988).]
As estimated by physician consultations, the incidence of symptomatic genital herpes is steadily increasing in the United States, developing among hundreds of thousands of Americans every year. [Sullender et al., J. Infect. Dis., 157(1): 164-167 (January 1988); Tapley et al. (eds.), The Columbia University College of Physicians and Surgeons Complete Home Medical Guide (1985).] The prevalence of asymptomatic HSV-2 infections has been difficult to determine because of the strong cross-neutralization between HSV-1 and HSV-2 and because of the high incidence of antibody to HSV-1 in the population. [Plummer, Cancer Res., 33:1469-1476 (June 1973).] As Lee et al. [J. Clin. Microbiol., 22(4): 641-644 at 643 (1985)] point out: "[A] serological assay that can detect HSV-2 antibodies would be of particular epidemiological assistance. However, because of the existence of many common antigens in HSV-1 and HSV-2, specificity of the assay has been a major problem." [Emphasis added; citation omitted.] Specificity of such an assay is important because of the implications of HSV-2 infections both at the epidemiological level, for example, the relation of genital herpes to cervical cancer, and at the individual level, for example, false-positive results can lead to great problems such as improper medical management for pregnant women or undue psychological trauma in patients and their consorts. [Lee et al., id. at 643.] The instant invention provides for a specific serological assay to differentiate accurately and definitively between HSV-1 and HSV-2 antibodies.
During the past 20 years, the incidence of neonatal HSV-2 infection has increased significantly, paralleling the increased incidence of genital HSV-2 infection in pregnant females--approximately a nine-fold increase from 1966 to 1979. [Hampar et al., U.S. Pat. No. 4,764,459 (Aug. 16, 1988).] A systemic HSV-2 infection in a newborn can cause serious problems, including blindness, neurological problems, mental retardation and even death. The major source of neonatal HSV-2 infection is via contact with the infected genital tract of the mother at the time of delivery. [Corey et al., Ann. Int. Med., 98:958-972 (1983).] Transmission of HSV-2 from mother to infant can occur during symptomatic or asymptomatic maternal infection. [Corey et al., id.] Studies have indicated that over 70% of infants with neonatal HSV-2 infection had mothers who were asymptomatic at the time of delivery. [Whitley et al., Pediatrics, 66:495-501 (1980).] The overall risk of neonatal infection has been estimated to be about 10% in women with primary or recurrent HSV-2 infection after 8 months' gestation and 40% if the HSV-2 virus is present at the time of delivery. [Douglas, "DNA Viruses: Herpetoviridae" in Principles and Practice of Infectious Diseases (2d Ed.) (Mandrell et al., eds. (1985).]
The recommended procedure for a pregnant woman suspected of harboring a HSV-2 infection is to perform weekly tissue culture confirmation tests to determine whether HSV-2 is being released into the birth canal. Such procedures are costly and, further, the tissue culture tests are limited by the timing of taking the samples, that is, only at certain points during the infectious cycle are samples containing live virus obtainable; for example, live virus is obtainable during the actively shedding stage in the cycle but not later when the vital vesicles are drying. [See Spruance et al. Infect. Inmnun. 36(3):907-910 (June 1982); and Spruance et al., N. Engl. J. Med. 297(2): 69-75 (Jul. 14, 1977).] Therefore, the use of tissue culture tests is limited to the high risk category of pregnant women who show either a) a history of recurrent genital HSV-2 infection, b) active disease during pregnancy, or c) sexual partners with proven HSV-2 infection. If an active infection is apparent, a cesarean delivery, with its associated risks, is performed as a protective measure for the baby. [Hampar et al., supra]. However, as noted above, most infants with neonatal infection due to HSV-2 are born to mothers with no history of genital herpes. Because asymptomatic intrapartum shedding of HSV-2 from the mother's cervical or vulval areas appear to be an important source of neonatal infection, a rapid, reliable and inexpensive serological screening test to identify pregnant women potentially harboring HSV-2 is needed. [Corey et al., supra]. This invention provides for such a screening test.
The serological assays of this invention are based upon recombinantly, synthetically or biologically produced proteins and polypeptides specific for HSV-2 antibodies. Such proteins and polypeptides are encoded by a unique DNA sequence, or fragments thereof, of the envelope protein, glycoprotein G (gG), of HSV-2, which sequence is not found in HSV-1. McGeoch et al. [J. Gen. Virol, 68:19-38 (1987)] identified the gene coding for gG in HSV-2, delineated its nucleotide and amino acid sequences, and pointed out (at p. 19) that the HSV-2 DNA contains "an extra sequence of about 1460 base pairs" which the HSV-1 gG gene does not have.
Both HSV-2 gG and HSV-1 gG have segments of 153 identical amino acids at their carboxyl-terminal end which contain their putative transmembrane anchor domain (McGeoch et al., id.). However, HSV-2 gG contains an additional segment of 487 unique amino acids which contain the putative type-2 specific epitopes observed with gG, and which are coded for by the extra "about 1460 base pairs" identified by McGeoch et al. Roizman et al., Virology, 133:242-247 (1984) and Marsden et al., J. Virol., 50(2): 547-554 (May 1984) independently discovered HSV-2 gG and developed monoclonal antibodies to it. Roizman et al. described two murine monoclonal antibodies that react with HSV-2 type-specific epitopes of HSV-2 gG and proved that gG was distinct from other HSV-2 envelope glycoproteins, namely, gB, gC and gD.
Use of the HSV-2 gG to detect HSV-2 type-specific antibodies has been reported by Lee et al. [J. Clin. Microbiol., 22(4): 641-644 (October 1985)], Sullender et al. [J. Inf. Dis., 157(1): 164-171 (January 1988)], and Ashley et al. [J. Clin. Microbiol., 26(4): 662-667 (April 1988)]. In each of these studies, immunoaffinity purified, native, full-length, glycosylated gG was employed. Since full-length gG was used, the assays were subject to cross-reactivity with HSV-1 antibodies in the test sera because of the commonality of certain domains in both HSV-1 and HSV-2 gG. Sullender et al. and Ashley et al. suggest the possible clinical use of the HSV-2 gG antibody assay in the diagnosis of genital infections and also in screening pregnant women. However, their assays, requiring the culturing of HSV-2, isolation of the virus and affinity purifying HSV-2 gG from viral lysate antigen preparations with monoclonal antibodies to HSV-2, are expensive to prepare and basically research tools at this time.
Hampar et al. [U.S. Pat. No. 4,764,459 (Aug. 16, 1988)] claims immunoassay methods for detecting antibodies to either HSV-1 or HSV-2 wherein the patients' sera are absorbed with heterologous virus-infected cell extracts to remove intertypic cross-reacting antibodies and then applied to microtiter plates containing the target antigens, either immunoaffinity purified HSV-1 glycoproteins (gC and/or gD) or HSV-2 glycoproteins (gD and/or gF).
Markoulatos et al. [European Pat. App. Pub. No. 263,025 (pub. Apr. 6, 1988)] discloses antigenic glycoprotein fractions of HSV-1 and HSV-2 (gC) and HSV-1 and HSV-2 (gD), purified from respectively infected cells, and claims their use to differentiate between HSV-1 and HSV-2 infections.
Su et al. [J. Virol., 62(10): 3668-3674 (October 1988) report expressing HSV-2 gG in a mammalian cell line. The gG expressed was full length and glycosylated.
Burke et al. [U.S. Pat. No. 4,618,578 (Oct. 21, 1986)] claims methods and compositions for recombinantly producing in yeast polypeptides which are immunologically cross-reactive with glycoprotein D (gD) of HSV-1 and HSV-2. Burke et al. state (at col. 2 lines 6-9) that the "[p]roduction of gD in a yeast host provides the advantages of high levels of expression and modification of the polypeptides not available with prokaryotic hosts . . . ."
Watson et al., [Science, 218:381-384 (Oct. 22, 1982)], report the expression of a HSV-1 gycoprotein D (gD) gene in Escherichia coli (E. coli). Watson et al. state that the fusion of the gD coding region with the E. coli lac promoter enabled them to synthesize a gD-related polypeptide, which when injected into rabbits elicited neutralizing antibody to both HSV-1 and HSV-2. Weis et al. [Nature, 302:72-74 (March 1983) report higher level of expression of gD in E. coli, wherein a hybrid gene encoding a chimaeric protein containing HSV-1 gD, bacteriophage lambda Cro and E. coli beta-galactosidase protein was constructed.
Berman et al. [European Pat. App. Pub. No. 139,417 (pub. Feb. 2 1989)] discloses the expression of HSV-1 glycoprotein D (gD) in Chinese hamster ovary cells (CHO). Claimed therein are vaccines against HSV-1 and HSV-2 comprising at least one glycoprotein of HSV-1 or HSV-2, preferably gD or gC.
Kino et al. [U.S. Pat. No. 4,661,349 (Apr. 28, 1987)] claims a HSV subunit vaccine effective against both HSV-1 and HSV-2 which comprises a highly purified native glycoprotein B (gB) common to both serotypes. Cohen et al. [U.S. Pat. No. 4,762,708 (Aug. 9, 1988)] discloses immunologically active preparations of purified, native HSV envelope glycoproteins, gD-1 and gD-2, useful in vaccines against HSV-1 and HSV-2.
At this time, the only commercially available means of differentially diagnosing a HSV-2 infection from a HSV-1 infection is by a monoclonal antibody-based tissue culture confirmation test which is relatively expensive compared to a blood test and time consuming, taking from at least 24 to 72 hours. Further, such tissue culture confirmation tests are limited because of the above-noted problems associated with obtaining tissue specimens with viable virus. Further, the tissue culture confirmation tests are prohibitively expensive for use in screening asymptomatic carriers of HSV-2. The instant invention provides a substantially cheaper, much quicker and non-time dependent method of serologically identifying HSV-2 type-specific antibodies.
Conventional wisdom in the immunochemistry art appears to consider native glycosylation patterns of antigens important to the conformational aspects of epitopes and necessary for serotype specificity. [See: Berman et al., Science, 222:524-527 at 525 (Nov. 4, 1983); Wilcox et al., J. Virol., 62(6): 1941-1947 (June 1988); Sugawara et al., J. Gen. Virol., 69 (pt. 3): 537-547 (March 1988); Caust et al., Arch. Virol., 96(3-4): 123-124 (1987); Hongo et al., Vaccine, 3(3 suppl.):223-226 (September 1985); Alexander et al., Science, 226 (4680): 1328-1330 (Dec. 14, 1984) Berman et al., EP 139,417 at pp. 1-2; but see: Glorioso et al., Virol., 126(1): 1-18 (Apr. 15, 1983) (wherein it is stated at p. 16: "Although carbohydrate does not appear to be essential for maintenance of antigenicity, it cannot be ruled out that the carbohydrate moieties may play an important role in protein conformation and that some antigenic determinant sites are formed as a consequence of protein secondary structure".] The instant invention controverts such conventional wisdom in that the recombinantly produced proteins and polypeptides of this invention which are type-specific for HSV-2 antibodies can be nonglycosylated, having been expressed in a prokaryotic host.